Fractionation of amino acid mixtures



March 25, 1952 E. J. ROBERTS 2,590,209

' FRACTIONATION OF AMINO ACID MIXTURES Original Filed March 4, 1947 13 Sheets-Sheet 2 Recycle Hydrolysate I" Mixture 2a k al 1 '4 zs a '5 FIG. 2. Amino. Cation l Acids Exchanger Step I: l absorbeq Bed 23 Loading (.Column 'l) l:, E l l 7 is I r 4 e 22 I f I 24 25 N waste file Recovered Recycle ale aste Water Acid Mixture Solution l I In Step 1110 9. 2 J

Neutrals. 32/ Acidics Zl 3 I 4 36 a, NW 33 a ar 31 7 \91 so 4' I I Basic Cation j' I Amino Exchanger p Acids Bed 26 Unloadmg Remain (Column *HLIE] (Neutrals 8t Acid|cs) I sei 44 42 I Waste Neutrals Neutrals Water 8lAcidics Acidics I & NH t F7 l J F IG. 3. INVENTOR To steplvz Elllott J. Roberts,

ATTORNEY March 25, 1952 ROBERTS 2,590,209

FRACTIONATION OF AMINd ACID MIXTURES Original Filed March 4, 1947 13 Sheets-sheet 3 f ,NH4 ./52 "a NH HCO 8r 0 2. 54 I l BOSiCS 5 Fl 4. I 4 62 Exhausted Exchange 29 G J L/ Exchanger Bed I 1 Left (ColumnH) I 5 I 56 I .s1 e 1 1I= i mmq B i 57 0 3 (Basics removed) 8 NH4 H003 Basics 59 l Evaporation H2 Basics 60 Br 00;

Fresh Recovered H 80 b Acid (from Stepl) 66 FIG. 5. Cation Re enerated 7 Exchange S'repDIa g Bed 28 ilegeneration (Co|umn H after StepIlI Waste I Regenerant 72 INVENTOR Elliott J. Roberts,

BY QM.Md-d&icw

ATTO R N EY March 25, 1952 E. J. ROBERTS 2,590,209

FRACTIONATION OF AMINO ACID MIXTURES Original Filed March 4, 1947 13 Sheets-Sheet 4 N .utrals 43 8| cidics P (from Stepll) 1 i FIG. 6.

i A 73 Acidics 2 Step IV: Remain Exchange Se 0 atin Bed a (Column ZL-Eg (to Recovery) I l FIG.7. Exchanger Anion Bed left in Exchange l regenerated Bed Step state. (Co|umn 2) fi] Removing I a 9| 94 i f a}; a W NH4+salts of 92 NH +salts ACidiCS \93 +NH4OH 95 1 I To StepVI for I f 2' L INVENTOR El ltott J. Roberts, WWW

ATTORNEY March 25, 1952 Original Filed March 4, 1947 H2804 IN E. J. ROBERTS FRACTIONATION OF AMINO ACID MIXTURES NH S alts, and acidics is Sheets-Sheet 5 Mother Liquors IOO Exhausted Exchanger left Waste Water Cation Exchange Bed (Column 3) NH Salts of Acidics (from Step'V) Cation Exchange Bed (Column 3) Liberating in HI Acidics Crystallizer Centrifuge Mixed Crystalline Acidics Water Step VI R e generation after Step VI To Waste Water Step VI NH +Salts 8 Acidics FIG. 8.

INVENTOR Elliott J. Roberts,

BY v

ATTORNEY March 25, 1952 E. J. ROBERTS FRACTIONATION OF AMINO ACID MIXTURES l3 Sheets-Sheet 8 Original Filed March 4, 1947 V cozoauem 2 coz om 9625 2 2280 o m tw 5.2m wa m cmtom J E itm m 2 388 c mo .65 zoom 0 n scam 8...:

h m m w n N INVENTOR Elliott J. Roberts,

ATTORNEY March 25, 1952 J. ROBERTS FRAcTIONA Iofi' OF AMINO ACID MIXTURES Original Filed March 4, 1947 13 Sheets-Shet 9 INVENTOR E/I/bJFJPaber/s ATTORNEY March 25, 19 52 E. J. ROBERTS FRACTIONATION 0F AMINO ACID MIXTURES 13 Sheets-Sheet 10 Original Filed March 4, 1947 March 25, 1952 E. J. ROBERTS 2,590,209

FRACTIONATION OF AMINO ACID MIXTURES Original Filed March 4, 1947 15 Sheets-Sheet l1 672,01 Un/oad/ng:

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INVENTOR ATTORNEY March 25, 1952 E. J. ROBERTS 2,590,209

'FRACTIONATION OF AMINO ACID MIXTURES Original Filed March 4, 1947 13 Sheets-Sheet l3 impure Am/na Acid M/Lrfures Cwl'on fa 017 a Bea {hm/1701?; Janfam/nanfs ATTORNEY tion whereby the acidics are separated from the neutrals, and this will be called Separating. The resulting fractions are herein also called the secondary fractions.

This Separating is accomplished by passing the fraction or solution that contains substantiallyonly the acidics and the neutrals in mixture through a bed of anion exchange material the selective action of which, under certain environmental conditions, allows the neutrals to pass as efliuent from the anion bed, but holds the acidics sorbed by the grains of the anion exchange material.

The next step is to remove from the anion bed the acidics residual therein, which step is herein called Removing and comprises treating the anion bed with an alkali solution, for example the hydroxide of ammonium or sodium, although the former is preferred because of its lower cost. The resulting eiiiuent solution contains the acidics fraction, for example glutamic and aspartic acids, but they are in the form of respective salts, in this case ammoniumor sodium glutamate and ammoniumor sodium aspartate (as the case may be, depending upon which hydroxide is used). It is proposed to liberate the acids as such by passing the solution through a bed of cation exchange material which operates in the H-ion cycle.

Thus this invention involves the steps of Loading, Unloading, Stripping, Separating, Removing, and Liberating.

Loading is the accumulation of all three groups of amino acids in the H-ion regenerated cation bed (column No. 1).

Unloading is the joint removal from that bed of the neutrals and the acidics.

Stripping is the removal from the cation exchange bed of the basics remaining therein after the neutrals and the acidics have first been jointly removed from that bed.

Separating is the fractionation of the neutrals from the acidics (column No. 2).

Removing is the relieving of the anion exchange bed of the acidics remaining therein after the neutrals have been fractionated therefrom.

Liberating is the reclaiming of the acidics as such from their salts (column No. 3).

Thereafter follows the recovery of these acidics in crystalline form.

In the fractionation of the different groups of amino acids, the control of pH is important, indeed critical if uncontaminated fractions are to be obtained. This will be gone into in detail as this specification proceeds, and indeed so will other refinements of this invention. Other features of the invention reside in the utilization of intermediate or contaminated fractions; the recovery of acid for re-use; the prevention of crystallization of acidics in the anion bed; and still other features that will appear as this specification proceeds.

It is noteworthy with respect to the conduct of the process according to this invention that in the acidic amino compounds the acidic amino acid molecule has only one basic radical but two acidic radicals, which structure renders the molecule preponderantly acidic; the neutral amino acids have in each molecule one basic. as well as one acid radical tending to balance and neutralize each other and thus rendering the molecule as such substantially neutral; the basic amino acids have in each molecule two basic radicals but only one acid radical, which structure renders the molecule preponderantly basic.

In other words, it is the additional acidor basic radical that renders the respective amino acid either more strongly acid or more strongly basic as the case may be.

That is, pH-characteristics of the respective acidic-, neutral-, and basic amino compounds are substantiated in and by the following tabulation (Table A) of pH values.

Complete proteins contain the three groups (neutral, acidic, and basic) of amino acids representatives of each of which are listed in the following tabulation:

TABLE A pH of Dilute Solutions Name P H d ure y ro- Acid chloride New A. Neutral Amino Acids (Nutritional) average pH 5.0 to 6.0:

Leucino 6. 0 1 1 11 Isoleucine. 6.0 1 1 11 Serine 5. 7 1 1 11 Thre0nine no data 1 1 11 Prolinc 6. 3 1 1 11 Hydroxyproline. 5. 7 1 1 11 Tyrosinc 5.7 1 1 9.6 Tryptophane 5. 9 1 1 11 Phenylalanine. 5. 5 1 1 11 Cystine 5.1 1 1 11 Methionine 5. 7 1 1 11 B. Acidic Amino Acids (Tasteimparting) average pH 3.0:

Aspartic Acid... 2. 8 1 1 0. 6 3.2 1 1 7.0

Arginine 10.8 1 5.6 11

1 Average pH 1.0. 2 Average pH 4.0.

This tabulation lists 19 of 21 known amino acids divisible into the above three groups and according to the pH of their aqueous solutions and the characteristic groupings influencing the pH. The neutral amino acids, in pure water solution, exhibit a pH of around 6.0. The acidic amino acids carry an extra COOH-group in the radical R and this acidic group causes the pH of their solutions to be around 3.0. The basic amino acids have an extra NH or NHz in the radical R, which basic group causes the pH to be on the alkaline side from 7.5 to 10.8. The tabulation presents not only the pH of the pure amino acid solution, but also the pH of the hydrochloride and of the Na-salt. These figures serve to bring out characteristic differences in the behavior of the groups although full titration curves of each are necessary to obtain a complete picture of the behavior characteristics.

With respect to primary separation, the pH of th hydrochloride solutions may explain the behavior of the groups on the cation exchange material. The basic amino acid hydrochlorides have a pH of 3.9 or greater while both the neutral and acidic amino acid hydrochlorides register a pH of less than 1.0. Thus at a pH of around 4.0 the basic amino acids will exist almost completely as cations and be held by the cation exchange material while the other two groups willbe almost entirely in the non-cationic form and therefore not be held by the cation exchange material. A separation is therefore possible.

With respect to secondary separation, the fact that pure solutions of the acidics have sufficient acid strength to give a pH of around 3 While the neutrals givea pH of 5.0-6.0 enables one to make a-separation of these two groups with an anion exchange material if no foreign cation are present:

The;problem of separating the basicfrom the neutral-and acidic amino acids by way of the cation exchange bed is not as simple as'might appear from the above, and likewise the problem ofseparating' the acidics'from the neutrals by way ofthe anion exchange bed.

One of the difliculties is that, unless the mixture of acidics and neutrals fed to the anion bed is free from cations, the separation will be'incomplete. For example,if an amount of Na+ or NHd-ions equivalent to 1% and the acidics is present, that amount of acidics willpass into the neutrals fraction, thus causing not only tantamount loss' of: acidics but also a proportionate contamination of the neutrals fraction by that amount of acidics and of Na+- or NH'i 'iOnS. Thepresent invention therefore provides that the. feed solution supplied to the anion exchange bed bei'substantially free from cations.

This invention teaches how to produce in primary separation this cation-free feed solution for the anion'exchange bed of the secondaryseparation operation. Accordingly, an efiiuent mixture of neutrals and acidics containing the equivalent of less than 1% of the acidics present is obtained. and control may be exercised whereby 0.1% is obtainable.

The pH of the efiluent from the cation exchange -bed is markedly influenced by the presence of cationsy hence pH measurements on the effluent serve as a basis for control.

If the electrodes of the pH indicator are placed in-the' bottom section of the bed, or if a small stream of solution is bled from a point somewhat above the bottom and run through the pH indicator, the pH indications will warn the operator of 'the approach of the cation breakthrough so that he may prevent undue contamination of a desired :efiluent fraction.

With'respect to the conduct of the operation of the: various exchange treatment steps of this process;ithe invention also provides that certain cuts be made between displaced void water and pregnant solution flowing from the bed at the beginning ofa step, as well as between the pregnant solution and the excess rinse water at the end of the step. An additional amount of barren solution that follows the efiluent' of void water from an exchange bed in certain steps can be sent to waste with a consequent reduction or elimination of the evaporation load.

According to one feature one fraction comprising, the'a'ci'dicand the neutral-, and another fraction comprising the basic amino acids are obtained from a mixture by Unloading the acidicsand the neutrals fraction from' the cation bed with'a solution substantially containing cations of auvolatile' salt along with anions which are noncontaminating with respect to the amino acids, for example OH-, glutamate-, aspartate: HCOr, 003 and then Stripping the basics fraction from the cation bed with a solution of the volatile salt, for. example ammonium carbonate or bicarbonate with the addition of CO2.

Another feature to effect the recovery ofan acidic and a neutral amino acid'fracticn per se from a inixture'provides for passing the mixture through. a bed of alkali-regenerated anion exchange-materialwhereby the acidic amino acids aresselectively. retained in the bed while the neutralsfara passed off. as efiluent, andv thenzalkalis 6? regenerating the. exchange bed to remove-the acidics assaltstain the resulting efiluentsolution.

According to anotherffeature'the acidic amino acids are liberated or reconstituted from. their' salts, such as glutamate or aspartataby passing the'effiuent solution: from the anion exchange bed through a hydrogen-ion regenerated bed of cation exchange material.

Another feature lies in purifying; acidic and neutral amino acids in a solution containingcontaminating cations.

Still another feature lies in'purifying' a solution. mixture of acidic andneutral amino acids orfree-- ing it'from' basic amino acids contained in the mixture.

tities of all groups as well as with contaminant cations.

A non-acid unloading solution of alkaline reaction havingnon-contaminating anions, such as NH4OH, is then passed'through the" cation exchange bed for effecting the primaryseparation' of the'group of basic amino compounds from the" groups'of acidicandneutral amino compoundsby selectively displacingthem from the bed with a solution of -NH4OH;.but not thebasics which resist displacement by that solution and are:held

residual on and by the bed.

The selective displacementof the acidandneutral amino compounds is explainable as follows:

The combination of the cation exchange mate rial with the acidic'andneutral amino'acids'may. be consideredxas" a salt in which the." exchange. material represents'the anionicpart while'the amino acid represents the cationicoribasicpart'. Whereasxweak bases'are displaceablefrom their salts by strongerbases, therefore the acidicand the=neutra1 amino acidsbeing butfeebly' basic are readily displaceable' by. the more strongly basic unloading chemicalsuch as'NHiOH." By

contrast, theba'sic amino acids'because' of their much greater'basicity' are. very much more re sistant'to such displacement and therefore remain" residual in the bed.

As the. resultant eflluent solution containing selectively displaced acidic and neutral amino" duction fraction, is collected. containing. the' (taste-imparting) acidics as: well as the'r(nu tritional) neutrals in the form of their acids and substantially free from contaminant cationsas well as from NHd-ions. Theuse' of." an alkali; for example NHrOH, as the. unloading'solution proposed by this invention furnishes an*exceptional criterion for rendering the fractionation accurate, that istwith respect to a desired'idegree of purity and relatively high concentration of the production fraction, for the reason that it provides a characteristic NHU-breakthrough in a charted curve representing characteristic guide points in the pH-change of the resultantefiluent solution. The. qualities in the production'frac tion to purity. andconcentration are important ioarendering eflicient the secondary separation; namely the separation of the a'cldics" from the neutrals in-the production fraction-of the'mixture;

The: residual; (nutritional) basic amino: compounds' are stripped. from the cationexchange bed- 1 by means of a: strippingsolutionlbff a salt andmore preferably. of? axvolatilessalt, n'amely ae carbonate or a bicarbonate of ammonium, it being notable that the cation of this stripping solution is of the same kind as that of the preceding hydroxide unloading solution of NHiOI-I. At any rate the residual basics are thus being stripped from the cation exchange bed by means of a salt solution whereas the acidics and neutrals had been unloaded from the bed by means of an alkaline solution.

Next, the production fraction representing a relatively cation-free and relatively concentrated solution of acidics and neutrals now in the form of their acids having resulted from the primary separation, is subjected to the secondary separation whereby to isolate (taste-imparting) acidics from (nutritional) neutrals, this in consideration of the fact that the acidics are much more strongly acid than are the neutrals. In fact, the neutrals are so weakly acid that the anion exchange material employed will not to any significant degree intercept and hold them, Whereas the acidics because of their greater acidity are intercepted and held by this material.

This secondary separation as between the acidics and the neutrals takes place as the cation free production fraction of' their mixture passes through a bed of alkali-regenerated anion exchange material with this bed retaining the acidics while rejecting the neutrals, the latter passing substantially pure from the bed in the resulting effluent solution.

The retained acidics are removed from the anion exchange bed by means of an alkali solution such as NHiOl-I being passed through the bed and producing an effluent solution containing the removed acidics as alkali salts. An intermediate efiluent portion or fraction containing NI-Lfi-salts of the acidics substantially free from NH4OH is collected as a production fraction to be subjected to a treatment whereby these alkali salts are converted or reconstituted to form the acidic amino acids proper.

That is to say, the production fraction of these alkali salts of the acidics is subjected to what is herein termed the liberating operation by being contacted and treated with acid-regenerated cation exchange material which reconstitutes these salts so as to form the desired acidic amino acids proper in which form they pass from a bed of such material in the resulting efiluent solution.

This effect of reconstituting the acidics as acidic amino acids as such will be more clearly understood as follows:

When the alkali salts of the acidic amino acids pass through a freshly acid-regenerated bed of cation exchange material, both the salt cations (NHi and the acidic amino acids of the salts are retained by the bed. But the exchange material of the bed discriminates against the amino acids in favor of the salt cations, the material having greater affinity for the cations than for the amino acids, so that the salt cations will occupy the infiuent end portion of the bed, while amino acids will occupy a portion of the bed subjacent to the salt cations. Because of this preference of the exchange material more and more of the bed becomes saturated with cations while tute and can be intercepted in an efiiuent production fraction in which they are contained at relatively high concentration and pure. These 8 qualities of purity and concentration of the production fraction free from NHi-ions are attainable with the aid of an exceptional criterion which presents itself in the Nl-Ii-breakthrough with the attendant sharp change of the pH of the efiluent solution of which the production frac-- tion is a part.

In order to preclude undesired crystallization of the acidic amino acids from a solution thus relatively highly concentrated, the feed solution of alkali salts of the acidic amino acids is passed to and through the bed at a sufiiciently high temperature level.

With the aid of the pH-criterion just mentioned the intermediate production fraction of this effluent solution is collected for and may be subjected to crystallization treatment for yieldin acidic amino acids in crystalline form. Howover, mother liquor resulting from the crystallization operation is utilized by being passed through an acid-regenerated cation exchange bed in a subsequent operation leading towards the reconstituting of the acidic amino acids such as above set forth.

This invention presents a process for treating an aqueous solution mixture of amino acids or protein hydrolysate so that a substantially full and direct recovery is made of each of the respective amino fractions per se, namely, of the acidics, the neutrals, and the basics, each substantially pure.

In summary, this invention calls for first loading all amino acids indiscriminately onto an acidregenerated cation exchange bed thereby separating the amino acids from their carrier solution. then selectively unloading the acidicand the neutral amino acids combined by means of ammonium hydroxide, and thereafter stripping from the bed the residual basic amino acids by means of ammonium carbonate or -bicarbonate.

A characteristic in the conduct of the process comprising this combination of treatment steps lies in collecting a first fraction or portion of the unloading effluent solution by the criterion of the abrupt increase or break in the rate of pH- change when the NH4 -iOI1S break through, and collecting a second fraction or portion of the efiiuent solution obtained subsequent to such break; the first fraction contains the acidics and the neutrals significantly pure and thus suited to be directly processed as in a bed of anion exchange material for separating the acidics from the neutrals with the acidics being retained by the bed so they can be liberated from the bed pure by alkali regeneration of the bed; the second eflluent fraction contains along with acidics and neutrals some of the basics together with unspent NHi and this fraction is employed to help in a subsequent unloading operation.

Even though the unloading solution and the stripping solution each have their specific characteristics to effect unloading and stripping respectively as conceived by this invention, in order that full recovery may be had of the acidics and of the neutrals in a pure state, it is necessary to accept a degree of admixture of basics to the acidics and neutrals which occurs past the NH4+- breakthrough point and such admixture is to be dealt with in the manner according to the concept of this invention. That is to say, the mixture of acidics, neutrals, basics and NH4+ contained in the second unloading efiiuent fraction is employed to serve in a subsequent unloading, whereas the acidics and neutrals in the first collected effluent fraction are significantly pure and 9 can be separated from one another as is. In this way, the acidics and neutrals are fully unloaded. from the bed and are obtainedeach isolated and pure, namely uncontaminated by either basics or by NH4+. that there be no acidics and neutrals left on the bed at the time of stripping the basics, yet that no basics will appear admixed 'in either the acidics or in the neutrals.

In loading, the starting solution or protein hydrolysate is passed through a bed of acid-regenerated cation exchange material whereby there is effected a separation of all the amino compounds from its carrier solution by indiscriminately loading the acidics, neutrals, and

basics onto the bed, preliminary to their being selectively recovered directly and fully therefrom in a pure state.

In unloading, the unloading solution (ammonium hydroxide) or base ammonia with a pH above 7.0, being but slightly ionizedacts'by themechanism of displacement. That is to say, the combination of acidics and neutrals with the exchange material behaves like salt of very weak bases; from such a combination the acidics and .neutrals are .readily displaced by the stronger base NH4OH. That is, by displacement weak bases are rendered into their non-cationic form in which they are not held by the exchange material and are eluted while the stronger base passes into its cationic for-m andis held by the exchange material in place of the eluted weak bases. However, this mechanism ofv displacement leaves the basics substantially unaffected except to some extent after the NH4+-breakthrough whensome basics and un-utilizedNHv -ions appear in mixture with acidics and neutrals'in the efiluent solution. Thus the effluent solution collected in a first fraction (with NI-IU-breakthrough as a criterion) contains unloaded acidics and neutrals in a pure state, and collected in a second fraction contains unloaded acidics and neutrals in mixture with some basics and with 'un-utilized NHU. The first or pure fraction may be sent to a bed of alkali-regenerated anion exchange material which'retains the acidics and passes the neutrals, the retained acidics to be liberated by alkali regeneration. Notably, the

second or mixed'fraction is employed to help in' a' subsequent unloading operation wherein the basic amino acids being more stronglybasicithan the neutral and acidic amino acids, act as unloading reagent in conjunction with un-utilized 'NH4+ to help elute'acidic and neutral amino acids from the bed.

In stripping, ammonium carbonate or -bicarbonate is used as astripping solution which is highlyionized presenting highconcentration of available cations to act effectively by way of direct ion-exchange as distinct from the mechanism of displacement, so'as to have the basics yield as ammonium salts although in mixture withvolatilizable and removable NH4OH in the 'efiluent. characterand ellect from theunloading solution .which..is substantially non-ionized and has Slittle Thus :the stripping solution differs in or secondaryeffect upon the basics on the'bed. In brief, this invention presents a process which effects the separation of an aqueous solution mix- .ture of amino acids contained ina protein hydrolysate into groups .of acidic-, neutral-, and basic amino acids respectivelyby treatment with This mixture or protein whereby it is passed through a bed of acidvregenerated cation exchange material for ab- Thus the invention provides sorbing thereon the amino acids indiscriminately from the solution while impurity anions pass in the efiluent from the bed. Thereupon the acidicand the neutral amino acids are selectively unloaded by displacing them from their combination with the exchange material by passing through the bed an unloading solution comprising ammonium hydroxide as a base to effect converting the acidicand the neutral amino acids but substantially not the more strongly held basic amino acids into non-cationic form in whichthey are eluted from the exchange mate- .rial whereby the material is left containing basic amino acids along with NHfi-cations while the acidicand the neutral amino acids are obtained in the eiiluent as displaced free amino acids. Meanwhile the resulting efiluent solution from this cation exchange bed is collected in a first fraction up to a point defined substantially by a point of abrupt increase in the pH-change of the effluent solution, which first fraction contains acidicand neutral amino acids substantially pure, namely uncontaminated by NH4+- cations. Following this first fraction there is collected a second fraction of the effluent solution containing substantially the remainder of the acidic and the neutral amino acids although contaminated by NHi -cations from the unloading solution and also mixed with a minor quantity of the basic amino acids displaced by the unloading solution while a major quantity thereof remains on the bed.

Thenfollows the stripping step which effects the stripping of the basic amino acids from the .bedbypassing through the bed a stripping solution comprising as stripping reagent a salt of a .group consisting of ammonium carbonate and a subsequent unloading operation whereby acidic and neutral amino acids in said fraction are recovered while NH4+ as well as the basic amino acids therein act as unloading agents.

The invention is illustrated diagrammatically or symbolically in the accompanying drawings" in which various steps of the process of this invention are indicated.

Fig. l is a treatment diagram illustrative. of the major steps of this invention involving the recovery per se of the acidics-, the neutrals-,

and the basics fraction;

v.Fig. 2 shows Step I, namely, Loading the cation bed.

Fig. 3 shows Step II, namely, the Unloading from the cation bed of the neutrals and the acidics jointly;

Fig. 4 shows Step III, namely, the Stripping of the basics from the cation bed;

Fig. 5 shows Step III namely, regeneration of the exhausted cationbed after it has been unloaded and stripped;

Fig. 6 shows Step IV, namely, Separating .of the neutrals from the acidics remaining in the anion exchange bed;

Fig. 7 shows Step V, namely, Removingthe remaining acidics from the anion exchange bed;

Fig. 8 shows Step VI, namely, Liberating in the second cation exchange bed the acidics from the contaminated solution in which form they issue from the anion exchange bed;

Fig. 9 shows Step VI namely, the regeneration of the second cation exchange bed;

Figs. 10 to 16 represent momentary chemical conditions of the three ion exchange beds;

Fig. 17 represents an operational control graph for the Unloading step.

Fig. 18 represents an operational control graph of pH-criteria for conducting the Liberating Step.

Fig. 19 illustrates the conduct of the Liberating Step in terms of conditions of the cation exchange bed.

Fig. 20 represents Steps I to VI in terms of Ion Exchange Equilibrium Equations.

Fig. 21 presents diagrammatically the overall concept of the process in terms of the Primary and the Secondary Separation of the amino compounds.

Fig. 22 presents the overall concept of Fig. *21 although in terms of ion exchange stations.

The Fig. 2l-fiowsheet conveys broadly the relationship between the Primary separation and the Secondary separation of the respective amino acids; namely primary separation of a mixture (A) of contaminants (salts), acidics, neutrals, and basics into cation-free acidics plus neutrals as one primary fraction (B) and basics as the other primary fraction (C), and secondary separation of the mixture of cation-free acidics and neutrals into acidics as one secondary fraction (D) and neutrals as the other secondary fraction (E). Thus the largely taste-imparting acidics such as glutamic and aspartic acids will have been separated from the largely nutritional neutrals and basics.

The Fig. 22- flowsheet, like Fig. 21, presents the overall relationship between the Primary and the Secondary separation of the respective amino acid groups, but implemented by ion-exchange stations; namely a 1st cation exchange bed for primary separation whereby a mixture of acidics and neutrals is separated from the basics, an anion exchange bed for secondary separation whereby acidics (although in the form of alkali salts) are separated from the neutrals, and a 2nd cation exchange bed whereby the acidics are liberatedor reconstituted from their alkali salts.

The operation according to the Fig. 1-fiowsheet while concerned with recovery of the acidics, the neutrals, and the basics as isolated fractions, is more explicit as to the character of the treatment steps than is the Fig. 22-flowsheet.

In Fig. 1 the starting material preferably has a high content of amino acids so by way of illustration there is herein proposed the use of hydrolyzed or acid-digested soy bean meal after oil has been extracted therefrom. The result is a hydrolysate rich in amino acids and wherein the environment of the acids has been changed so that their separation from their environment and from each other is rendered possible. Such hydrolysate or other solution of amino acids is passed through a bed or column of cation exchange material loading the same (Step I).

Thereupon the primary fractionation begins by passing suitable displacing solutions through the bed. One such solution displaces or unloads from the bed that primary fraction which includes the neutrals and acidics which pass as efiluent along one fiowpath from thebed (Step II) A portion of the efliuent is recycled in order to obtain this fractionneutrals .plus acidicssubstantially cation-free.

The alkaline unloading solution contains the cations of the same kind that are contained in the volatile salt that is being used in the stripping operation and non-contaminating anions such as OII, glutamatr, aspartate: HCOr, (303 Another such solution subsequently displaces or strips from the bed that fraction which includes the basics which have remained in the bed and which now pass as efiiuent along another fiowpath from the'bed (Step III), whereupon they may be subjected to treatment whereby they are recovered in concentrated form.

To effect the recovery of the basics by stripping, the solution is that of a volatile salt such as ammonium carbonate or bicarbonate.

In order to effect the secondary fractionation or separation as between the neutrals and the acidics, the primary fraction containing them in mixture is then flowed through a bed of anion exchange material with the result that the neutrals separate and emerge in the efiluent passing along one fiowpath from the bed (Step IV), while the acidics are left behind being held sorbed by the bed. A suitable displacing solution passed through the bed displaces the acidics thus being carried along another fiowpath in the effluent passing from the bed (Step V).

The two secondary fractions, that is the neutrals on the one hand, and the acidics on the other, may then be individually subjected to treatment whereby they are recovered in crystalline or concentrated form, or they may first be further fractionated.

The operation according to one embodiment of this invention is concerned solely with the recovery of the neutrals and of the acidics as fractions. Hence the solution to effect Unloading is defined as a non-acidic solution of noncontaminating anions such as OH: glutamate: aspartate HCO3 COa=. The subsequent steps of Separating (Step IV), Removing (Step V), and Liberating (Step VI) correspond to similarly identified steps in the Fig. 1 embodiment.

An unloading solution suited for the present purpose contains cations selected from the group consisting of ammonium, alkali metals, alkaline earth metals and basic amino acids, and noncontaminating anions. The non-contaminating anions for the present purpose are those which in the course of the process form water or which are Volatile and can, therefore, be readily eliminated, or which are amino acids, and which do not form insoluble precipitates with any of the cations present.

At present only one of the amino acids has commercial value in some other than the nutritional field, that is glutamic acid which in the form of its sodium salt is used widely as a fiavoring agent. While the other amino acids are of value nutritionally, only ten of them are at present considered essential for human subsistence. These are histidine, lysine, arginine, valine, leucine, isoleucine, threonine, phenylalanine, tryptophane, and methionine. Of these arginine does not appear to be essential in the adult, and histidine may be dispensable in humans.

Most proteins contain all of the amino acids in Table A in varying proportions. Gluten wheat is rich in glutamic acid and low in lysine. When glutamic acid is manufactured from gluten (which is at present one of the chief commercial sources) all of the other amino acids may be discarded since, due to their low lysine content, they are not worth purification. Most present- 13 day medical (nutritional) preparations of the amino acids are derived from casein or other milk proteins Casein contains the essential amino acids in desirable proportions and is readily obtained in a rather pure state which is highly desirable when using present-day methods of processing to produce the hydrolysate.

Soy bean meal (after extraction to remove oil) is a much cheaper source, containing nitrogen equivalent to 44% of protein, and 5.4% lysine based on 16% N according to Block, while casein contains 6.9%. It is one of-the objects of this invention to isolate glutamic acid from soy meal .and then to produce cheaply the nutritional amino acid mixture from a cheap source. By removing the acidic amino acids from soy meal hydrolysate, the lysine concentration in the finished concentrate should be as high as that made from casein. Again, if casein could be used as a cheap enough source, the removal therefrom of the acidics would increase the lysine concentration by 40%.

In order to separate the glutamic acid from the other amino acids it is necessary first to make a separation of the neutral and acidic amino acids from the basic amino acids and other bases. Thereafter the neutrals and acidics can be separated from each other. After the three groups have thus been isolated, the neutrals and basics can be combined for nutritional end uses, while the acidics could be processed to produce flavoring substances.

Proteins can be broken down into a mixture of amino acids by means of enzymes, alkalies, or strong acids. The invention underlying the present process of fractionating certain amino acids or amino acid groups from a mixture is concerned particularly with such a mixture or protein hydrolysate which has been produced by strongacid hydrolysis. Concentrations of hydrolyzing acids frequently employed in protein hydrolysis are, for example, 20% (6 N) HCl or 25% (6 N) H2804. As a net result the acid is not actually consumed by the hydrolysis reaction although the hydrochlorides or hydrosulfates of the amino acids are formed, the real purpose of the hydrolyzing acid being to introduce water into the bonds which tie up or interlock the amino acids .in the protein.

For the purpose of the present process of frac- 'tionating amino acids or amino acid groups it is advantageous to have the protein source as free from metallic cations as possible since their presence will make themselves felt in the later phases of the operation when they appear as a contamination in the basics fraction and must be dealt with by way of some suitable elimination treatment if the desired pure crystals of the basic amino acids are to be derived from this process. Among the impurities potassium, for

example, is commonly present in natural plant the capacity of the exchange bed with a given amount of recycle mixture and hence upon the economics of the process. I have found that the lower this ratio the higher is the bed capacity, and I therefore propose to use as little acid as possible in the hydrolysis of the natural protein, although sufiicient acid should be used toobtain complete hydrolysis along with humin ofv good filtering qualities. i

HCl and H2804 when used as hydrolyzing acids each present their individual advantages and disadvantages. That is to say, H01 as a hydrolyzing acid is difficult to work with and is relatively expensive although the quantity required is smaller than the required quantity of H2804. With HCl as the hydrolyzing acid, the eflluent from the cation exchange bed resulting from the loading phase (Step I) can be reused for hydrolysis after it has been subjected to a recovery treatment wherein most of the water is distilled off and .then a 15% to 20% HCl solution distilled and condensed for reuse in another protein hydrolysis.

H2804 as hydrolyzing acid is uneconomical to recover for reuse as a hydrolyzing agent, but for otherwise good economy I propose to recover and reuse a quantity thereof in the acid regeneration of the cation exchange bed.

In the hydrolysis operation a large excess of hydrolyzing acids appears to do no harm, but the minimum quantity is controlled by three factors:

(a) There should be enough liquid present in' the hydrolysate to produce a fluid mixture which is capable of being handled mechanically;

(b) There should be suflicient acidpresent to produce a humin residue (forming from the carbohydrate material present in the protein) that is readily filterable.

(c) It appears that there must be present at least one molecule of HCl and H2804 for each molecule of amino acid; hence, 2 equivalents of H2804 are required against 1 equivalent of HCl, this presumably because the second hydrogen ion of H2804 is weaker.

Relatively lower concentrations of hydrolyzing acid can be used if the above three factors (a'),

(b), and (c) are satisfied although a longer peabout 2.5 lbs of 6 N H2804 are required per pound of crude protein to satisfy conditions (a) and (b). However, where the protein is very pure this will not furnish sufficient H2804 to satisfy condition (0).

As a source of the amino acids, naturally occurring solutions of the same may be used; also enzymatically hydrolyzed solutions if such solu-- tions do not contain substances harmful to the exchange beds. Such naturally occurring solutions may include sugar juices derived from beet or cane.

Loading (Step I) .The term Loading connotes the absorbing of the amino acids from a hydrolysate on a cation exchange bed and recovering acid for reuse be it for hydrolysis or for regenerating purposes. In Fig. 2 there is diagrammatically illustrated the loading step or phase whereby the total mixture of amino acids is collected upon a bed of cation exchange material herein also briefly termed cation bed, from'the protein hydrolysate.

This leading phase comprises the sequential 

1. THE PROCESS FOR THE SEPARATION OF AN AQUEOUS SOLUTION MIXTURE OF AMINO ACIDS SUCH AS A PROTEIN HYDROLYSATE INTO GROUPS OF ACIDIC-, NEUTRAL-, AND BASIC-AMINO ACIDS RESPECTIVELY BY TREATMENT WITH ION EXCHANGE MATERIAL, CHARACTERIZED IN THAT THE MIXTURE IS PASSED THROUGH A BED OF ACID-REGENERATED CATION EXCHANGE MATERIAL FOR ABSORBING THEREON THE AMINO ACIDS INDISCRIMINATELY WHILE IMPURITY ANIONS PASS IN THE EFFUENT FROM THE BED, SELECTIVELY, UNLOADING THE ACIDIC AND NEUTRAL AMINO ACIDS BY DISPLACING THEM FROM THEIR COMBINATION WITH THE EXCHANGE MATERIAL BY PASSING THROUGH THE BED AN UNLOADING SOLUTION COMPRISING AMMONIUM HYDROXIDE AS A BASE TO EFFECT CONVERTING THE ACIDIC AND NEUTRAL AMINO ACIDS BUT SUBSTANTIALLY NOT THE MORE STRONGLY HELD BASIC AMINO ACIDS INTO NONCATIONIC FORM IN WHICH THEY ARE ELUTED FROM THE EXCHANGE MATERIAL WHEREBY THE MATERIAL IS LEFT CONTAINING BASICAMINO ACIDS ALONG WITH NH4+-CATIONS WHILE THE ACIDICS AND NEUTRAL AMINO ACIDS ARE ABTAINED IN THE EFFLUENT AS DISPLACED FREE AMINO ACIDS, MEANWHILE COLLECTING THE RESULTING EFFUENT SOLUTION IN A FIRST FRACTION UP TO A POINT DEFINED SUBSTANTIALLY BY A POINT OF ABRUPT INCREASE IN THE PHCHANGE OF THE EFFUENT SOLUTION, SAID FRACTION CONTAINING ACIDIC AND NEUTRAL AMINO ACIDS SUBSTANTIALLY PURE, NAMELY UNCONTAMINATED BY NH4+ CATIONS, AND IN A SECOND FRACTION CONTAINING SUBSTANTIALLY THE REMAINDER OF THE ACIDIC AND NEUTRAL AMINO ACIDS ALTHOUGH CONTAMINATED BY NH4+-CATIONS FROM THE UNLOADING SOLUTION AND ALSO MIXED WITH A MINOR QUANTITY OF THE BASICAMINO ACIDS DISPLACED BY THE UNLOADING SOLUTION WHILE A MAJOR QUANTITY THEREOF REMAINS ON THE BED, THEN PASSING THROUGH THE BED A STRIPPING SOLUTION COMPRISING AS STRIPPING REAGENT A SALT OF A GROUP CONSISTING OF AMMONIUM CARBONATE AND BICARBONATE PROVIDING A HIGH CONCENTRATION OF AMMONIUM CATIONS AS DISTINCT FROM THE BASE AMMONIA WHICH IS NOT HIGHLY IONIZED IN SOLUTION FOR EFFECTING IONIC EXCHANGE AS BETWEEN THE AMMONIUM CATIONS AND THE CATIONIC BASIC-AMINO ACIDS ON THE BED WHEREBY THERE RESULT IN THE EFFUENT THE BASIC-AMINO ACIDS AS SALTS TOGETHER WITH VOLATILE MOLECULES OF EXCESS STRIPPING REAGENT, SUCH STRIPPING REAGENT GEING SEPARABLE FROM THE BASICAMINO ACID SALTS BY VOLATILIZATION, AND EMPLOYING SAID SECOND FRACTION IN ADDITION TO SAID UNLOAD ING SOLUTION IN A SUBSEQUENT UNLOADING OPERATION WHEREBY ACIDIC NEUTRAL AMINO ACIDS, IN SAID FRACTION ARE RECOVERED WHILE NH4+ AS WELL AS THE BASIC AMINO THEREIN ACT AS UNLOADING AGENTS. 